Downregulation of miR-16 protects H9c2(2-1) cells against hypoxia/reoxygenation damage by targeting CIAPIN1 and regulating the NF-κB pathway

Abstract

The aim of the present study was to determine the function of microRNA-16 (miR-16) in myocardial hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and the possible mechanism underlying its involvement. An H/R model was constructed using H9c2(2-1) cells in vitro. The results of reverse transcription-quantitative PCR demonstrated that the expression levels of miR-16 were significantly upregulated in H9c2(2-1) cells in the H/R group compared with the sham group (1.53±0.09 vs. 1.0±0.08; P=0.0019). Cell Counting Kit-8 assays revealed that the relative proliferative ability of H9c2(2-1) cells was significantly decreased in the H/R + negative control (NC) group compared with the sham group (0.53±0.05 vs. 1.0±0.08; P=0.00005). Upregulation of miR-16 using miR-16 mimics further decreased the proliferative ability of cells (0.31±0.03 vs. 0.53±0.05; P=0.0097), whereas downregulation of miR-16 using an miR-16 inhibitor increased the proliferative ability of cells compared with the H/R+NC group (0.89±0.08 vs. 0.53±0.05; P=0.000385). Flow cytometric analysis found that the apoptotic rate of H9c2(2-1) cells was increased significantly following H/R compared with the sham group (25.86±2.62% vs. 9.29±0.82%, P=0.000014). Upregulation of miR-16 further increased the apoptotic rate (38.62±2.04% vs. 25.86±2.62%; P=0.000099), whereas downregulation of miR-16 decreased the apoptotic rate compared with the H/R+NC group (15.14±0.92% vs. 25.86±2.62%; P=0.000343). miR-16 directly bound to the 3'-untrans-lated region of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) and negatively modulated CIAPIN1 expression. Overexpression of CIAPIN1 reversed the changes in the expression of apoptosis-associated proteins caused by H/R. Western blot analysis revealed that the levels of phospho-(p-) nuclear factor-κB (NF-κB) and p-NF-κB inhibitor α (IκBα) were upregulated following H/R (1.82±0.11 vs. 1.0±0.08; P=0.000152; and 1.77±0.07 vs. 1.0±0.00; P=0.000024, respectively), and these changes were further enhanced when miR-16 expression levels were increased (3.10±0.14 vs. 1.82±0.11; P=0.000006; and 2.19±0.10 vs. 1.77±0.07; P=0.0017, respectively). Downregulation of miR-16 exhibited the opposite effect on p-NF-κB and p-IκBα expression levels. The present study illustrates that downregulation of miR-16 may protect against H/R-induced injury partially by targeting CIAPIN1 and the NF-κB signaling pathway.